Helena Bacha Lopes

Titanium With Nanotopography Induces Osteoblast Differentiation by Regulating Endogenous Bone Morphogenetic Protein Expression and Signaling Pathway.

We aimed at evaluating the effect of titanium (Ti) with nanotopography (Nano) on the endogenous expression of BMP‐2 and BMP‐4 and the relevance of this process to the nanotopography‐induced osteoblast differentiation. MC3T3‐E1 cells were grown on Nano and machined (Machined) Ti surfaces and the endogenous BMP‐2/4 expression and the effect of BMP receptor BMPR1A silencing in both osteoblast differentiation and expression of genes related to TGF‐β/BMP signaling were evaluated. Nano supported higher BMP‐2 gene and protein expression and upregulated the osteoblast differentiation compared with Machined Ti surface. The BMPR1A silencing inhibited the osteogenic potential induced by Nano Ti surface as indicated by reduced alkaline phosphatase (ALP), osteocalcin and RUNX2 gene expression, RUNX2 protein expression and ALP activity. In addition, the expression of genes related to TGF‐β/BMP signaling was deeply affected by BMPR1A‐silenced cells grown on Nano Ti surface. In conclusion, we have demonstrated for the first time that nanotopography induces osteoblast differentiation, at least in part, by upregulating the endogenous production of BMP‐2 and modulating BMP signaling pathway. J. Cell. Biochem. 117: 1718–1726, 2016.

Poly(vinylidene-trifluoroethylene)/barium titanate composite for in vivo support of bone formation

In this study, we evaluated the effect of poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane on in vivo bone formation. Rat calvarial bone defects were implanted with P(VDF-TrFE)/BT and polytetrafluoroethylene (PTFE) membranes, and at 4 and 8 weeks, histomorphometric and gene expression analyses were performed. A higher amount of bone formation was noticed on P(VDF-TrFE)/BT compared with PTFE. The gene expression of RUNX2, bone sialoprotein, osteocalcin, receptor activator of nuclear factor-kappa B ligand, and osteoprotegerin indicates that P(VDF-TrFE)/BT favored the osteoblast differentiation compared with PTFE. These results evidenced the benefits of using P(VDF-TrFE)/BT to promote new bone formation, which may represent a promising alternative to be employed in guided bone regeneration.

Effect of Surface Nanotopography on Bone Response to Titanium Implant

Clinical success of implant therapy is directly related to titanium (Ti) surface properties and the quality of bone tissue. The treatment of Ti implants with H2SO4/H2O2 is a feasible, reproducible, and low-cost technique to create surface nanotopography (Ti-Nano). As this nanotopography induces osteoblast differentiation, we hypothesized that it may affect bone response to Ti. Thus, this study was designed to evaluate the bone response to a machined Ti implant treated with H2SO4/H2O2 to generate Ti-Nano and to compare it with a commercially available microtopographic Ti implant (Ti-Porous). Implants were placed in rabbit tibias and evaluated after 2 and 6 weeks, and the bone tissue formed around them was assessed by microtomography to record bone volume, bone surface, specific bone surface, trabecular number, trabecular thickness, and trabecular separation. Undecalcified histological sections were used to determine the percentages of bone-to-implant contact, bone area formed between threads, and bone area formed in the mirror area. At the end of 6 weeks, the removal torque was evaluated using a digital torque gauge. The results showed bone formation in close contact with both Ti-Nano and Ti-Porous implants without relevant morphological and morphometric differences, in addition to a similar removal torque irrespective of surface topography. In conclusion, our results have shown that a simple and low-cost method using H2SO4/H2O2 is highly efficient for creating nanotopography on Ti surfaces, which elicits a similar bone response compared with microtopography presented in a commercially available Ti implant.

Participation of MicroRNA-34a and RANKL on bone repair induced by poly(vinylidene-trifluoroethylene)/barium titanate membrane

Abstract The poly(vinylidene-trifluoroethylene)/barium titanate (PVDF) membrane enhances in vitro osteoblast differentiation and in vivo bone repair. Here, we hypothesized that this higher bone repair could be also due to bone resorption inhibition mediated by a microRNA (miR)/RANKL circuit. To test our hypothesis, the large-scale miR expression of bone tissue grown on PVDF and polytetrafluoroethylene (PTFE) membranes was evaluated to identify potential RANKL-targeted miRs modulated by PVDF. The animal model used was rat calvarial defects implanted with either PVDF or PTFE. At 4 and 8 weeks, the bone tissue grown on membranes was submitted to a large-scale analysis of miRs by microarray. The expression of miR-34a and some of its targets, including RANKL, were evaluated by real-time polimerase chain reaction and osteoclast activity was detected by tartrate-resistant acid phosphatase (TRAP) staining. Among more than 250 miRs, twelve, including miR-34a, were simultaneously higher expressed (≥2 fold) at 4 and 8 weeks on PVDF. The higher expression of miR-34a was concomitant with a reduced expression of all its evaluated targets, including RANKL. Additionally, more TRAP-positive cells were observed in bone tissue grown on PTFE compared with PVDF in both time points. In conclusion, our results suggest that the higher bone formation induced by PVDF could be, at least in part, triggered by a miR-34a increase and RANKL decrease, which may inhibit osteoclast differentiation and activity, and bone resorption.

Association of mesenchymal stem cells and osteoblasts for bone repair.

AIM We tested the hypothesis that the association of bone marrow mesenchymal stem cells (MSCs) and osteoblasts (OBs) optimize bone repair. MATERIALS & METHODS MSCs were cultured in growth or osteogenic medium and seeded into gelatin sponge prior to implantation. Defects were created into rat calvariae and implanted with gelatin sponge without cells, with MSCs, with OBs and with association of MSCs and OBs. Histological analysis and micro-CT-based histomorphometry were carried out after 4 weeks. RESULTS Increased bone formation was observed in defects treated with cells and bone volume was greater in defects treated with either OBs or MSCs/OBs. CONCLUSION Association of MSCs and OBs did not increase the process of bone repair compared with cell-based therapy using either MSCs or OBs alone.

Participation of extracellular signal-regulated kinases 1/2 in osteoblast and adipocyte differentiation of mesenchymal stem cells grown on titanium surfaces

Osteoblasts and adipocytes coexist in the implantation site and affect the process of titanium (Ti) osseointegration. As extracellular signal-regulated kinases 1/2 (ERK1/2) are involved in osteogenesis and adipogenesis, the aim of our study was to investigate if the effects of Ti surface topography on osteoblast and adipocyte differentiation are modulated by ERK1/2. The experiments were conducted based on the effect of the ERK1/2 inhibitor, PD98059, on mesenchymal stem cells (MSCs) grown under osteogenic and adipogenic conditions on Ti with nanotopography (Ti-Nano) or on machined Ti (Ti-Machined). The results showed that, in general, ERK1/2 inhibition favored osteoblast and adipocyte differentiation of MSCs grown on Ti-Machined. In MSCs grown on Ti-Nano, ERK1/2 inhibition upregulated the expression of alkaline phosphatase and osteocalcin and reduced extracellular matrix mineralization. In terms of adipocyte differentiation, ERK1/2 inhibition elicited similar MSC responses to Ti-Nano and Ti-Machined, upregulating gene expression of adipocyte markers without affecting lipid accumulation. Our results indicate that, under osteogenic and adipogenic conditions, the responses of MSCs to Ti surface topography in terms of osteogenesis and adipogenesis are dependent on ERK1/2. Thus, a precise modulation of ERK1/2 expression and activity induced by surface topography could be a good strategy to drive the process of implant osseointegration.

MicroRNA 665 Regulates Dentinogenesis through MicroRNA-Mediated Silencing and Epigenetic Mechanisms

ABSTRACT Studies of proteins involved in microRNA (miRNA) processing, maturation, and silencing have indicated the importance of miRNAs in skeletogenesis, but the specific miRNAs involved in this process are incompletely defined. Here, we identified miRNA 665 (miR-665) as a potential repressor of odontoblast maturation. Studies with cultured cell lines and primary embryonic cells showed that miR-665 represses the expression of early and late odontoblast marker genes and stage-specific proteases involved in dentin maturation. Notably, miR-665 directly targeted Dlx3 mRNA and decreased Dlx3 expression. Furthermore, RNA-induced silencing complex (RISC) immunoprecipitation and biotin-labeled miR-665 pulldown studies identified Kat6a as another potential target of miR-665. KAT6A interacted physically and functionally with RUNX2, activating tissue-specific promoter activity and prompting odontoblast differentiation. Overexpression of miR-665 reduced the recruitment of KAT6A to Dspp and Dmp1 promoters and prevented KAT6A-induced chromatin remodeling, repressing gene transcription. Taken together, our results provide novel molecular evidence that miR-665 functions in an miRNA-epigenetic regulatory network to control dentinogenesis.

Selection of reference genes for quantitative real-time polymerase chain reaction studies in rat osteoblasts: ABUNA et al.

Quantitative real‐time polymerase chain reaction (qRT‐PCR) is a powerful tool to evaluate gene expression, but its accuracy depends on the choice and stability of the reference genes used for normalization. In this study, we aimed to identify reference genes for studies on osteoblasts derived from rat bone marrow mesenchymal stem cells (bone marrow osteoblasts), osteoblasts derived from newborn rat calvarial (calvarial osteoblasts), and rat osteosarcoma cell line UMR‐106. The osteoblast phenotype was characterized by ALP activity and extracellular matrix mineralization. Thirty‐one candidates for reference genes from a Taqman® array were assessed by qRT‐PCR, and their expressions were analyzed by five different approaches. The data showed that several of the most traditional reference genes, such as Actb and Gapdh, were inadequate for normalization and that the experimental conditions may affect gene stability. Eif2b1 was frequently identified among the best reference genes in bone marrow osteoblasts, calvarial osteoblasts, and UMR‐106 osteoblasts. Selected stable and unstable reference genes were used to normalize the gene expression of Runx2, Alp, and Oc. The data showed statistically significant differences in the expression of these genes depending on the stability of the reference gene used for normalization, creating a bias that may induce incorrect assumptions in terms of osteoblast characterization of these cells. In conclusion, our study indicates that a rigorous selection of reference genes is a key step in qRT‐PCR studies in osteoblasts to generate precise and reliable data.

Effect of cell therapy with allogeneic osteoblasts on bone repair of rat calvaria defects

BACKGROUND AIMS Regenerative medicine strategies based on cell therapy are considered a promising approach to repair bone defects. The aims of this study were to evaluate the effect of subculturing on the osteogenic potential of osteoblasts derived from newborn rat calvaria and the effect of these osteoblasts on bone repair of rat calvaria defects. METHODS Cells were obtained from 50 newborn rat calvaria, and primary osteoblasts (OB) were compared with first passage (OB-P1) in terms of osteogenic potential by assaying cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization and gene expression of the osteoblastic markers RUNX2, ALP, osteocalcin and bone sialoprotein. Then, 5-mm calvaria defects were created in 24 Wistar rats, and after 2 weeks, they were locally injected with 50 µL of phosphate-buffered saline containing either 5 × 106 osteoblasts (OB-P1, n = 12) or no cells (control, n = 12). Four weeks post-injection, the bone formation was evaluated by micro-computed tomography and histological analyses. Data were compared by analysis of variance, followed by the Student-Newman-Keulss test or Students t-test (P ≤ 0.05). RESULTS OB-P1 showed high proliferation and ALP activity, and despite the reduced gene expression of osteoblastic markers and extracellular matrix mineralization compared with OB, they displayed osteogenic potential, being a good choice for injection into calvaria defects. The micro-tomographic and histological data showed that defects treated with OB-P1 presented higher bone formation compared with control defects. DISCUSSION Our results indicate that cells derived from newborn rat calvaria retain osteoblastic characteristics after subculturing and that these osteoblasts stimulate bone repair in a rat calvaria defect model.

Surgical Treatment of Fracture in Atrophic Jaw

Introduction:Mandible fractures are common injuries in the facial bones. It has a varied etiology, especially automobile accidents, bicycle accidents, motorcycle accidents and assaults. Objective: This study aims to illustrate a case of a patient with bilateral atrophic mandibular fractures treated surgically. Clinical case:male patient, 48 years old, victim an accident at work causing bilateral mandibular body fractures treated by internal rigid fixation with titanium plates and screws with the making of access submandibular Risdon bilaterally. Conclusion: The authors present a discussion of the controversy involved in this type of treatment and showing the advantages of transcutaneous approach for the treatment of mandibular fractures.